Supplementary Table S3 from Antixenograft tumor activity of a humanized anti-insulin-like growth factor-I receptor monoclonal antibody is associated with decreased AKT activation and glucose uptake

Supplementary Fig. S2 from Antixenograft tumor activity of a humanized anti-insulin-like growth factor-I receptor monoclonal antibody is associated with decreased AKT activation and glucose uptake

Supplementary Fig. S1 from Antixenograft tumor activity of a humanized anti-insulin-like growth factor-I receptor monoclonal antibody is associated with decreased AKT activation and glucose uptake

The blood-brain barrier (BBB) poses a major challenge for developing effective antibody therapies for neurological diseases. Using transcriptomic and proteomic profiling, we searched for proteins in mouse brain endothelial cells (BECs) that could potentially be exploited to transport antibodies across the BBB. Due to their limited protein abundance, neither antibodies against literature-identified targets nor BBB-enriched proteins identified by microarray facilitated significant antibody brain uptake. Using proteomic analysis of isolated mouse BECs, we identified multiple highly expressed proteins, including basigin, Glut1, and CD98hc. Antibodies to each of these targets were significantly enriched in the brain after administration in vivo. In particular, antibodies against CD98hc showed robust accumulation in brain after systemic dosing, and a significant pharmacodynamic response as measured by brain Aβ reduction. The discovery of CD98hc as a robust receptor-mediated transcytosis pathway for antibody delivery to the brain expands the current approaches available for enhancing brain uptake of therapeutic antibodies.

ntibody-mediated inhibition of R-spondin-3 in colorectal tumours decreases tumour growth and promotes differentiation—these effects are associated with a decrease in expression of genes associated with stem-cell function.

Bispecific antibodies engineered to both bind to the primate transferrin receptor and inhibit β-secretase are taken up by the nonhuman primate brain and reduce brain β-amyloid.

Melanocytes uniquely express specialized genes required for pigment formation, some of which are maintained following their transformation to melanoma. Here we exploit this property to selectively target melanoma with an antibody drug conjugate (ADC) specific to PMEL17, the product of the SILV pigment-forming gene. We describe new PMEL17 antibodies that detect the endogenous protein. These antibodies help define the secretory fate of PMEL17 and demonstrate its utility as an ADC target. Although newly synthesized PMEL17 is ultimately routed to the melanosome, we find substantial amounts accessible to our antibodies at the cell surface that undergo internalization and routing to a LAMP1-enriched, lysosome-related organelle. Accordingly, an ADC reactive with PMEL17 exhibits target-dependent tumor cell killing in vitro and in vivo.

It has been recently reported that treatment with an anti-placenta growth factor (PlGF) antibody inhibits metastasis and primary tumor growth. Here we show that, although anti-PlGF treatment inhibited wound healing, extravasation of B16F10 cells, and growth of a tumor engineered to overexpress the PlGF receptor (VEGFR-1), neutralization of PlGF using four novel blocking antibodies had no significant effect on tumor angiogenesis in 15 models. Also, genetic ablation of the tyrosine kinase domain of VEGFR-1 in the host did not result in growth inhibition of the anti-VEGF-A sensitive or resistant tumors tested. Furthermore, combination of anti-PlGF with anti-VEGF-A antibodies did not result in greater antitumor efficacy than anti-VEGF-A monotherapy. In conclusion, our data argue against an important role of PlGF during primary tumor growth in most models and suggest that clinical evaluation of anti-PlGF antibodies may be challenging.

Tim-4 is a phosphatidylserine (PS) receptor that is expressed on various macrophage subsets. It mediates phagocytosis of apoptotic cells by peritoneal macrophages. The in vivo functions of Tim-4 in phagocytosis and immune responses, however, are still unclear. In this study, we show that Tim-4 quickly forms punctate caps on contact with apoptotic cells, in contrast to its normal diffused expression on the surface of phagocytes. Despite its expression in marginal zone and tingible body macrophages, Tim-4 deficiency only minimally affects outcomes of several acute immune challenges, including the trapping of apoptotic cells in the marginal zone, the clearance apoptotic cells by tingible body macrophages, and the formation of germinal centers and elicitation of antibody responses against sheep red blood cells (SRBCs). In addition, Tim-4(-/-) resident peritoneal macrophages (rPMs) phagocytose necrotic cells and other opsonized targets normally. However, their ability to bind and engulf apoptotic cells is significantly compromised both in vitro and in vivo. Most importantly, Tim-4 deficiency results in increased cellularity in the peritoneum. Resting rPMs produce higher TNF-alpha in culture. Their response to LPS, on the contrary, is dampened. Our data support an indispensible role of Tim-4 in maintaining the homeostasis of rPMs.

Antibody-based therapeutic approaches have revolutionized the treatment of non- Hodgkin's lymphomas (NHL) as well as other hematological malignancies. However, given the biological and clinical heterogeneity of the diseases, a large variability in clinical response has been observed. Currently, patients with refractory or relapsed lymphomas have limited therapeutic options, and therefore the need to develop effective new treatments remains urgent.

Antibody-based therapeutic approaches have revolutionized the treatment of non-Hodgkin's lymphomas (NHL) as well as other hematological malignancies. However, a large variability in clinical response has been observed and patients with refractory or relapsed lymphomas have limited therapeutic options. Antibody drug conjugates (ADC) may prove valuable in this respect. The B cell surface antigen, CD79b, is an attractive target for this approach as it is rapidly internalized upon antibody engagement and has an appropriate expression pattern, being expressed only on normal and malignant B-cells. Anti-CD79b-MCC-DM1 is an ADC consisting of a chimeric monoclonal anti-CD79b antibody, 10D10, conjugated via a thioether linker to a cytotoxic drug, DM1 (a potent tubulin polymerization inhibitor). To investigate the pharmacodynamics (PD), pharmacokinetics (PK), and tolerability of 10D10-MCC-DM1 in nonhuman primates, we administered 10D10-MCC-DM1 or unconjugated antibody 10D10 to cynomolgus monkeys. 10D10-MCC-DM1 and 10D10 resulted in sustained depletion of B-cells and B cell subsets in blood, with more substantial depletion in the group given 10D10-MCC-DM1. These findings correlated well with the depletion of B-cells observed in lymphoid tissues (bone marrow, lymph nodes, and spleen). Both 10D10-MCC-DM1 and 10D10 were well tolerated. Taken together, these preclinical data suggest that targeting CD79b with ADCs may provide a safe and effective therapy for B-cell malignancies in humans.

Antibodies directed against B cells are in use for the treatment of non-Hodgkin's lymphoma and autoimmune disorders. The B-cell-restricted surface antigen CD79b, a signaling component of the B-cell receptor, has been shown as a promising antibody target in mouse efficacy models of systemic lupus erythematosus. Anti-CD79b antibody-drug conjugates (ADC), cytotoxic drugs linked through specialized chemical linkers to antibodies, are effective in mouse xenograft models of non-Hodgkin's lymphoma. We were interested in evaluating the systemic effects of anti-CD79b antibodies and ADCs in normal animals as a step toward the development of these molecules as therapeutics. As we were unable to identify any cell surface binding anti-human CD79b antibodies that were cross-reactive to other species, we developed an antibody to cynomolgus monkey (Macaca fascicularis) CD79b (anti-cyCD79b). The anti-cynomolgus antibody, anti-cyCD79b (10D10), and the maytansine (tubulin inhibitor)-conjugated ADC, anti-cyCD79b (10D10)-MCC-DM1, were administered to cynomolgus monkeys at approximately 30 mg/kg (6,000 microg DM1/m(2)) for two doses 3 weeks apart. Anti-cyCD79b and anti-cyCD79b-MCC-DM1 resulted in peripheral blood B-cell depletion of approximately 65% and approximately 94%, respectively. In addition, anti-cyCD79b-MCC-DM1 resulted in near-complete absence of splenic germinal centers, an observation supporting an effect on dividing B cells. Both molecules were well tolerated, with minimal findings for the antibody and findings for the ADC limited to the lymphoid and hematopoietic systems, liver, and peripheral nerves. These preclinical data suggest that targeting CD79b with antibodies or ADCs may provide safe and effective therapies for B-cell malignancies and autoimmune diseases.

Abstract The insulin-like growth factor (IGF) system consists of two ligands (IGF-I and IGF-II), which both signal through IGF-I receptor (IGF-IR) to stimulate proliferation and inhibit apoptosis, with activity contributing to malignant growth of many types of human cancers. We have developed a humanized, affinity-matured anti-human IGF-IR monoclonal antibody (h10H5), which binds with high affinity and specificity to the extracellular domain. h10H5 inhibits IGF-IR-mediated signaling by blocking IGF-I and IGF-II binding and by inducing cell surface receptor down-regulation via internalization and degradation, with the extracellular and intracellular domains of IGF-IR being differentially affected by the proteasomal and lysosomal inhibitors. In vitro, h10H5 exhibits antiproliferative effects on cancer cell lines. In vivo, h10H5 shows single-agent antitumor efficacy in human SK-N-AS neuroblastoma and SW527 breast cancer xenograft models and even greater efficacy in combination with the chemotherapeutic agent docetaxel or an anti–vascular endothelial growth factor antibody. Antitumor activity of h10H5 is associated with decreased AKT activation and glucose uptake and a 316-gene transcription profile with significant changes involving DNA metabolic and cell cycle machineries. These data support the clinical testing of h10H5 as a biotherapeutic for IGF-IR-dependent human tumors and furthermore illustrate a new method of monitoring its activity noninvasively in vivo via 2-fluoro-2-deoxy-d-glucose-positron emission tomography imaging. [Mol Cancer Ther 2008;7(9):2599–608]

Targeting cytotoxic drugs to cancer cells using antibody-drug conjugates (ADCs), particularly those with stable linkers between the drug and the antibody, could be an effective cancer treatment with low toxicity. However, for stable-linker ADCs to be effective they must be internalized and degraded, limiting potential targets to surface antigens that are trafficked to lysosomes. CD79a and CD79b comprise the hetrodimeric signaling component of the B-cell receptor (BCR), and are attractive targets for the use of ADCs since they are B-cell specific, expressed in non-Hodgkin’s lymphomas (NHLs), and trafficked to a lysosomal-like compartment as part of antigen presentation. We show here that the stablelinker ADCs anti-CD79b-MCC-DM1 and anti-CD79b-MC-MMAF are capable of target-dependent killing of NHL cell lines in vitro. Further, these two ADCs are equally effective at low doses in xenograft models of follicular, mantle cell, and Burkitt’s lymphomas, even though several of these cell lines express relatively low levels of CD79b in vivo. In addition, we demonstrate that anti-CD79b ADCs were more effective than anti-CD79a ADCs and that, as hypothesized, antiCD79b antibodies down-regulated surface BCR and were trafficked to the lysosomal-like compartment MIIC. These results suggest that anti-CD79b-MCCDM1 and anti-CD79b-MC-MMAF are promising therapeutics for the treatment of NHL.

Blood line VEGF, or vascular endothelial growth factor, is the best-characterized inducer of tumour angiogenesis, and the blockade of VEGF has become an important tool in cancer therapy. But VEGF blockade is not effective against all tumours, so the search for alternative approaches continues. Two groups this week report that one such alternative could be blockade of Dll4, Delta-like ligand 4. This transmembrane molecule is part of the Notch signalling pathway. It was known to be essential for normal development of blood vessels in the embryo: the new work shows that it is also required for tumour angiogenesis. It may be a viable — and potentially well tolerated — alternative in patients with solid tumours that are resistant to anti-VEGF therapy.

A new family of Ig domain receptors referred to as the immune receptor translocation-associated (IRTA) proteins, FcR homologs (FcRHs) or FcR-like that are expressed in lymphoid cells has been recently described. RNA expression analysis suggests that FcRH1-5/IRTA1-5 are expressed exclusively in subsets of the B-cell compartment. We generated mAbs to FcRH1-5/IRTA1-5 and examined their protein expression pattern in normal tissue and in chronic lymphocytic leukemia (CLL) cells. Our data indicated that FcRH1-5/IRTA1-5 were expressed in B-cell sub-populations; however, in some cases, the protein was not expressed in the same B-cell populations as suggested by the RNA expression analysis. FcRH1/IRTA5 was expressed throughout the B-cell lineage starting at the pro-B-cell stage but was down-regulated in plasma cells. FcRH2/IRTA4 was expressed preferentially in memory B cells. FcRH3/IRTA3 was expressed at low levels in naive, germinal center (GC) and memory B cells but was also expressed in NK cells. FcRH4/IRTA1 was expressed in a sub-population of memory B cells associated with mucosal tissue. FcRH5/IRTA2 was expressed in mature B cells and memory B cells and down-regulated in GC cells and, unlike all other B-cell-specific markers, maintained its expression in plasma cells from tonsil, spleen and bone marrow. We examined the expression of FcRH1-5/IRTA1-5 on the surface of CLL cells and found a similar pattern of expression on CLL cells as in the normal mature B cells, except for FcRH3/IRTA3 which was up-regulated in CLL.

Analysis of human colorectal cancer specimens revealed overexpression of the EphB2 receptor tyrosine kinase. Monoclonal antibodies (MAbs) to extracellular sequence of EphB2 were raised and tested for activity against colorectal cancer cells. One of the MAbs, 2H9, effectively blocked the interaction of ephB2 with ephrin ligands and inhibited the resulting autophosphorylation of the receptor. However, this antibody did not affect the proliferation of cancer cells expressing ephB2. Immunocytochemical analysis revealed rapid internalization of the MAb 2H9 on binding ephB2, suggesting that target-dependent cell killing could be achieved with an antibody-drug conjugate. When MAb 2H9 was conjugated to monomethylauristatin E through a cathepsin B-cleavable linker, it specifically killed ephB2-expressing cancer cells in vitro and in vivo. Our results suggest that ephB2 is an attractive target for immunoconjugate cancer therapy.

We conducted an expression analysis of prostate stem cell antigen (PSCA) in normal urogenital tissues, benign prostatic hyperplasia (n = 21), prostatic intraepithelial neoplasia (n = 33), and primary, (n = 137) and metastatic (n = 42) prostate adenocarcinoma, using isotopic in situ hybridization on tissue microarrays. In normal prostate, we observe PSCA expression in the terminally differentiated, secretory epithelium; strong expression was also seen in normal urothelium. Forty-eight percent of primary and 64% of metastatic prostatic adenocarcinomas expressed PSCA RNA. Our studies did not confirm a positive correlation between level of PSCA RNA expression and high Gleason grade. We characterized monoclonal anti-PSCA antibodies that recognize PSCA expressed on the surface of live cells, are efficiently internalized after antigen recognition, and kill tumor cells in vitro in an antigen-specific fashion upon conjugation with maytansinoid. Unconjugated anti-PSCA antibodies demonstrated efficacy against PSCA-positive tumors by delaying progressive tumor growth in vivo. Maytansinoid-conjugated antibodies caused complete regression of established tumors in a large proportion of animals. Our results strongly suggest that maytansinoid-conjugated anti-PSCA monoclonal antibodies should be evaluated as a therapeutic modality for patients with advanced prostate cancer.